Subtyping of hepatitis C virus with high resolution mass spectrometry

Clinical Mass Spectrometry 4–5 (2017) 19–24

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Subtyping of hepatitis C virus with high resolution mass spectrometry Reaz Uddin, Kevin M. Downard

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MARK

University of New South Wales, Sydney, Australia

A R T I C L E I N F O

A B S T R A C T

Keywords: Subtyping Hepatitis Virus Proteotyping Mass spectrometry

A proteotyping approach using high resolution mass spectrometry has been applied, for the first time, to subtype the hepatitis C virus based upon detection of one or more signature peptides derived from the E1 and E2 envelope glycoproteins. These signature peptides represent conserved peptide segments within these proteins for particular subtypes of the virus that are found to be unique in mass when compared with the theoretical masses for all peptide segments of translated HCV proteins within a specifically constructed database. The successful application of the approach to three different subtypes of the virus (i.e., 1a, 1b and 2b) is demonstrated for protein and whole virus proteolytic digests. The approach has the potential to replace existing PCR-based subtyping by offering a more direct and cost comparable strategy that is not challenged by mixed infection scenarios.

1. Introduction Hepatitis C virus (HCV) can cause severe inflammation of the liver with long-term complications, including cirrhosis and liver cancer [1]. The virus is primarily transmitted via blood exposure, although it can also be passed by sexual contact. There are six major genotypes of HCV. Genotypes 1–3 are widely distributed in developed countries, while the other genotypes are geographically localized [2,3]. Genotype 4 occurs in the Middle East, Egypt, and central Africa, while genotypes 5 and 6 are present almost exclusively in South Africa and Southeast Asia, respectively. In Australia, the predominant genotypes are 1 (representing some 54% of cases) and 3 (37% of cases), while genotype 2 accounts for fewer than 5% of cases [4]. Each genotype can be further subdivided into subtypes, denoted as 1a, 1b, 1c, et cetera. Correct genotype identification is clinically important as it allows customization of the dose and duration of antiviral drug therapies [5,6]. HCV contains a single-stranded, positive-sense RNA genome. It is approximately 9.6 kb in length with one open reading frame (ORF) that encodes a single polyprotein of approximately 3000 amino acids. This polyprotein is cleaved by both host and viral proteases to yield ten individual viral proteins denoted core C, envelope E1 and E2, polymerase p7, and non-structural NS2, NS3, NS4A, NS4B, NS5A and NS5B. The envelope glycoproteins E1 and E2 facilitate virus entry into the cell to initiate infection [7], yet the genes that encode these glycoproteins are the most variable of the group in terms of their nucleotide sequence. Variations in gene, and hence protein, sequence can alter a virus’ antigenic properties thereby affording the virus an avenue to escape

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neutralizing antibodies [8]. Genotyping involves the amplification of a segment of the genome by reverse transcription polymerase chain reaction (RT-PCR). Most commonly, a 5′-untranslated region (5′-UTR) is amplified since it is highly conserved and has a well-characterized set of polymorphisms that enable a strain’s genotype to be predicted [9]. The less-conserved core envelope [10] and NS5B [11] regions allow strains to be subtyped. The amplification products can be detected by probe hybridization assays [12], restriction site changes [13] and direct sequencing with detection of the amplicons using fluorescence [14] or even mass spectrometry [15]. Due to wide genetic diversity, the primers used in PCRbased methods have varying affinity to their sequence targets. This necessitates the use of multiple primers along the genome, including within variable areas, and these primers often need to be subtypespecific and even strain-specific. Additionally, sequencing of the RNA genome of HCV is not practical unless the samples contain high viral loads [16]. We have developed and applied a more direct and rapid proteotyping approach that exploits the capabilities of high resolution mass spectrometers to characterize viruses and other microorganisms [17]. It enables strains to be typed [18], subtyped [19–21] and their lineages [22] assessed through single-ion detection of a conserved signature peptide, or peptides, following whole virus digestion with a site-specific protease. Signature peptides represent segments of a viral protein that are conserved in sequence and are unique in mass with respect to all other proteolytic peptide segments across all viral proteins for all

Corresponding author at: Infectious Disease Responses Laboratory, POWCS, Medicine, University of New South Wales, Sydney, NSW 2052, Australia. E-mail address: [email protected] (K.M. Downard).

http://dx.doi.org/10.1016/j.clinms.2017.08.003 Received 21 June 2017; Received in revised form 20 August 2017; Accepted 21 August 2017 Available online 24 August 2017 2376-9998/ © 2017 Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).

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R. Uddin, K.M. Downard

characterized strains. The approach has been applied to study respiratory viruses, in the case of both influenza [18–22] and parainfluenza [23] virus, and can correctly type, subtype and establish the lineage of viral strains from clinical specimens [24]. The approach is amenable to parallel sample processing and analysis allowing mass spectra for several hundred specimens to be acquired in minutes. A computer algorithm (FluTyper) [25] has been developed to automatically interpret these spectra to type viruses. Here, we apply the proteotyping approach, for the first time, to an oncovirus in the form of hepatitis C virus. Signature peptides derived from the E1 and E2 envelope glycoproteins have been identified that enable viruses to be subtyped based upon their detection in a high resolution mass spectrum. 2. Materials and methods 2.1. Signature peptide determination for subtypes of HCV Full-length sequences for the E1 and E2 envelope proteins from confirmed subtypes of HCV were sourced from the European HCV (euHCV) [26] and Uniprot databases. Redundant sequences were removed and those for each of the different subtypes were concatenated into a single file and aligned using the Clustal Omega algorithm [27]. The aligned sequences were inputted into the FluAlign algorithm [19] to establish a consensus sequence and residue frequency at each position. The masses for tryptic peptide segments across the consensus sequence with the highest sequence conservation were assessed using the FluGest algorithm to establish signature (Po > 0.90) and indicator peptides (Po > 0.70) for each subtype. These were determined to be unique in mass (within 5 ppm) compared to all other tryptic peptides predicted in silico for a HCV viral protein database of 96,987 sequences comprised of all known sequences for unprocessed polyprotein, core protein, envelope glycoprotein E1, envelope glycoprotein E2, and various non-structural proteins of HCV.

Fig. 1. SDS-PAGE gel of deglycosylated recombinant E1 and E2 proteins.

20 hours at 37 °C and a portion of the proteolysis mixture was diluted with a MALDI matrix solution, as described below. 2.4. High resolution MALDI-MS of tryptic peptides E1/2 proteins Reconstituted peptides (∼1.6 μg/μL in 3 μL water) were diluted in a 5 mg/mL solution of α-cyano-4-hydroxycinnamic acid (8 μL containing 50% by volume acetonitrile and 0.1% trifluoroacetic acid). The sample solution was mixed at a 1:1 ratio (by volume) with matrix solution and 1 μL of the mixture was spotted in duplicate onto a Bruker MTP Anchorchip 400/384 TF plate (Bruker Daltonics, Billerica, MA, USA). High-resolution FT-ICR mass spectra were obtained on a 7T SolariX or Apex Qe instrument (Bruker Daltonics, Billerica, MA, USA) in the positive ion-mode, as previously described [19]. Spectra were acquired from an average of 5 to 10 scans, using a broadband excitation, and plotted over a m/z range up to 5000. The monoisotopic masses associated with E1/2 derived peptides were identified by matching their predicted values with those theoretically generated using the PeptideMass algorithm (http://web.expasy.org/peptide_mass/).

2.2. Deglycosylation and tryptic digestion of recombinant HCV envelope glycoproteins Recombinant envelope glycoproteins E1 (subtype 1b) and E2 (subtype 1a) of hepatitis C virus strains, each expressed with a polyhistidine tag at the N-terminus, were purchased from Sino Biological Inc. (Beijing, China) with predicted molecular weights of approximately 19 and 32 kDa respectively. Given their extensive glycosylation, the glycoproteins were deglycosylated with 1.2 units of recombinant peptide-N-glycosidase F (PNGaseF) (Roche Diagnostics, Sydney, Australia) and the process verified by SDS-PAGE (Fig. 1). The gel plugs for the lowest molecular bands (B2) were destained, treated with dithiothreitol and iodoacetoamide to reduce and alkylate the cysteine residues, and digested overnight at 37 °C with sequencing-grade, modified porcine trypsin endoproteinase (Promega Corporation, Sydney, Australia) in 25 mM ammonium bicarbonate according to a published method [26]. The extracted peptides were completely dried in a Labconco Centrivap (Labconco, Kansas City, MO, USA) prior to MALDI-MS analysis.

3. Results Non-redundant full-length sequences for the E1 and E2 envelope proteins of known HCV subtype were obtained from both the European HCV (euHCV) [27] and Uniprot databases and aligned with Clustal Omega [28] (Tables 1 and 2). Those subtypes with 20 or fewer nonredundant sequences were not considered further, since no reliable consensus sequence could be derived from such a limited dataset.

2.3. Whole HCV digest Table 1 Number of full-length sequences of E1 for each HCV subtype.*

An uncharacterised human strain of the hepatitis C virus was obtained from Zeptometrix (Buffalo, New York, USA) via Diagnostic Technology (Belrose, Sydney, Australia). After sonication of the stock solution, 50 μL was treated with 5 units of PNGaseF and heated for 24 hours at 37 °C. A separate portion of 10 μL was added to 9.5 μL of 50 mM ammonium acetate buffer (pH 6.7), containing 5 μL of mercaptoethanol, and incubated for 2 hours at 55 °C. Subsequently, the same volume of iodoacetamide was added and the incubation process repeated. The solution was then incubated with trypsin (2 μg) for

Subtype

euHCV

Uniprot

Total

Non-redundant

1a 1b 2b 3a

224 358 26 28

0 131 0 1

224 489 26 29

195 457 22 28

* Subtypes with few non-redundant sequences (≤20) for analysis were not considered for this study.

20

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(Supplementary Table S1). When this larger peptide alone, or in conjunction with the smaller signature peptide, is detected in the high resolution mass spectrum of an envelope protein derived from hepatitis C virus, or within a whole hepatitis C virus digest, the strains can be confidently assigned to the 1a subtype. Two indicators were determined for E1 proteins of the 1b subtype (Table 3). The indicator peptide at m/z 566.2932 is found in a large number of polyprotein sequences containing the E1 protein (i.e., 681), as is to be expected, and shares a mass within 5 ppm of only 8 other peptides (Supplementary Table S2); it is a reliable indicator of strains of the 1b subtype. The identity of the subtype is further enhanced when the second indicator peptide, at m/z 1301.7035, is also detected in the same spectrum. This indicator shares a common mass (within 5 ppm) with a few peptides among 92 strains derived from the NS3 structural protein (Supplementary Table S2). The high resolution mass spectrum for the tryptic digest of E1 envelope protein is shown in Fig. 2. Ions derived from peptide segments comprising 100 residues of a total of 149 were identified. These represent 67% sequence coverage across the protein. Among them is a 1b subtype indicator peptide at m/z 1358.7921 (Table 3) corresponding to residues 108–119 of the E1 protein (of m/z 1301.7035) in which the cysteine residue at position 108 is carbamidomethylated. Based upon the detection of this peptide ion alone the originating strain can be confidently determined to be subtype 1b. Similar confidence is shared in the identification of strains of the 2b subtype using the single-ion detection proteotyping strategy. The two indicator peptides (at m/z 502.2984 and 1357.7296) and one signature peptide (at m/z 1266.8031) (Table 3) share masses (within 5 ppm) with fewer than 10 other peptides derived from different proteins (see Supplementary Table S3). The same is true for the two indicators that identify strains of the 3a subtype: m/z 603.3249 and 1038.5579 (Table 3). The largest number of hits occurred for the polyprotein or partial polyproteins containing the E1 protein (Supplementary Table S4). Peptide segments with similar mass (within 5 ppm) are evident for other unrelated proteins in 11 or fewer characterised strains. In the same manner as that described for E1, signatures and indicators for the E2 envelope protein were identified across the major subtypes of HCV (Table 4) where sufficient E2 sequence data was available (Table 2). Peptides at m/z 1047.5040 and 1073.5197 were indicative of a 1a or 1b subtype, while peptides at m/z 960.4534 (1a subtype only) or 946.4628 (1b subtype only) were used to distinguish between the two. Ions detected at m/z 750.3087 and/or 1057.5248 were diagnostic of a strain of the 3a subtype. FluGest results for these indicator and signature peptides are shown in Supplementary Tables S5–S7. Note that no peptides were identified in the HCV protein database that shared a common mass (within 5 ppm) with the E2 indicator peptide at m/z 750.3087 (subtype 3a).

Table 2 Number of full-length sequences of E2 for each HCV subtype.* Subtype

euHCV

Uniprot

Total

Non-redundant

1a 1b 3a

219 332 28

82 65 559

301 397 587

271 370 396

* Subtypes with few non-redundant sequences (≤20) for analysis were not considered for this study.

The FluAlign algorithm [19] was employed to establish a consensus sequence for both proteins across all of the remaining subtypes and the frequency of each residue (denoted Ps or Pc at the C-terminal cleavage site) within tryptic peptide segments, generated in silico, were determined. Those with a Po value (where Po = Ps × Pc) that exceeded 0.90, or was between 0.70 and 0.90, were assigned as putative signature (s) or indicator (i) peptides, respectively (see Table 3 for E1 envelope protein). The theoretical masses for these peptides were compared with those generated from the in silico digestion of all proteins within the constructed HCV protein database. Those with values that differed from all others by less than or equal to 5 ppm were established as signature or indicator peptides. Analysis of the sequence and mass data for E1 proteins, of the 1a subtype, revealed a single signature peptide comprised of residues 60–63 (Table 3). This peptide segment had a theoretical monoisotopic mass for its protonated ions of m/z 565.3093. Its value was within 5 ppm of only six other tryptic peptide segments derived from the protein sequences, as shown in Table S1. Among those with the most hits were those that share a common sequence (i.e., YQVR) within 229 of all polyprotein sequences in the HCV database. This was expected since the unprocessed full-length polyprotein contains a segment that comprises the envelope E1 protein. A second segment of the same mass also arises within the 587 sequences for NS3 non-structural protein of strains of subtype 3a and 4a. This sequence (i.e., GGIYR) differs from YQVR in that it contains two glycine residues and an isoleucine residue with the same combined nominal residue mass (i.e., 227) compared to a glutamine and valine residue (Q + V), such that the peptides share a similar monoisotopic mass (within 5 ppm) (see Supplementary Table S1). This segment also occurs in 92 polyprotein sequences, including those that contain the E1 protein of the 1a subtype. The presence of the GGIYR peptide in a significant number of nonstructural and polyprotein sequences precludes the peptide YQVR from being a reliable independent signature for subtype 1a. However, 13 or fewer E1 proteins of other subtypes contain the same sequence (i..e, VLVVLLLFAGVDA), or a rearranged or common form, and thus mass (i.e., m/z 1328.8189), as the subtype 1a indicator peptide Table 3 Subtype specific signature and indicator peptides for HCV envelope glycoprotein E1. Subtype

Residues

Sequence

m/z [M+H]+ monoisotopic*

Ps

Pc

Po

Signature/indicator

1a 1a 1b 1b

60–63 239–251 1–4 108–119

YQVR VLVVLLLFAGVDA YEVR CWVALTPTLAAR

0.9295 0.6883** 0.8023 0.8176

0.9949 1 0.9581 0.9781

0.9247 0.6883* 0.7687 0.7997

s i** i i

2b 2b 2b

1–4 180–192 47–58

VEVR VIAILLLVAGVDA CWIQVTPNVAVK

0.8244 0.9091 0.8182

1 1 0.8636

0.8244 0.9091 0.7066

i s i

3a 3a

60–63 118–127

LEWR VIAILLLVAGVDA

565.3093 1328.8189 566.2932 1301.7035 (1358.7249) 502.2984 1266.8031 1357.7296 (1414.7511) 603.3249 1038.5579

0.8007 0.8302

1 0.9643

0.8007 0.8006

i i

The frequency of each residue (denoted Ps and Pc at the C-terminal cleavage site) was multiplied (Po = Ps × Pc) across each tryptic peptide segment. * Mass values in brackets denote peptide mass where cysteine residues are modified in the form of carbamidomethyl-cysteine. ** Denotes peptide was retained as an indicator since its Po value has a rounded value of 0.69, only just below the 0.70 cut-off.

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Fig. 2. MALDI mass spectrum of the tryptic digest products of recombinant E1 protein of the 1b subtype.

Table 4 Subtype signature and indicator peptides for HCV envelope glycoprotein E2. Subtype

Residues

Sequence

m/z [M+H]+ monoisotopic*

Ps

Pc

Po

Signature/indicator

1a 1a 1a

206–213 248–256 214–223

HPEATYSR MYVGGVEHR CGSGPWITPR

0.8821 0.8758 0.7824

1 0.9890 0.9963

0.8821 0.8661 0.7796

i i i

1b 1b 1b

207–214 249–257 215–224

HPEATYTK MYVGGVEHR CGSGPWLTPR

0.7761 0.9170 0.9517

0.9973 1 1

0.7740 0.9170 0.9517

i s s

3a

281–286

CDIEDR

0.7604

0.9824

0.7469

i

3a

226–235

CGAGPWLTPR

960.4534 1047.5040 1073.5197 (1130.5411) 946.4628 1047.5040 1073.5197 (1130.5411) 750.3087 (807.3301) 1057.5247 (1114.5462)

0.9529

0.9924

0.9457

s

The frequency of each residue (denoted Ps and Pc at the C-terminal cleavage site) was multiplied (Po = Ps × Pc) across each tryptic peptide segment. * Mass values in brackets denote peptide mass where cysteine residues are modified in the form of carbamidomethyl-cysteine.

to sequence and subtype HCV strains, currently available genotyping methods have limitations both in terms of their routine practice [16] and subtyping accuracy [29]. Even when a “deep” sequencing HCV subtyping strategy was employed [29], almost 4% of the strains cannot be subtyped, compared with 16% for the same 114 clinical specimens using conventional genotyping. The steps involved to achieve these results are not insignificant. RNA extraction is followed by reverse transcription using a pair of upstream and downstream primers and the production of amplified products confirmed by agarose gel electrophoresis. Aliquots of the PCR product are subjected to Sanger sequencing or ultradeep pyrosequencing (UDPS) subtyping, the latter employing a universal primer pair. The product of this amplification is often purified using magnetic beads and quantified using a fluorometric titration approach. Variability across the HCV genome constitutes the main difficulty in developing a robust technique where primers can anneal for every viral subtype. Furthermore, since genomic diversity is not consistent across different sections of the genome1, there is neither standardization in the positioning of primers, nor the primers used, across different laboratories [16]. Subtypes are identified based upon

The MALDI mass spectrum of the tryptic digestion products of deglycosylated E2 protein of the 1a subtype is shown in Fig. 3. Despite the spectrum exhibiting ions for fewer peptide segments, 86 of the 278 residues (or 31% coverage), it contains the signature peptide for strains of the 1a and 1b subtype comprising E2 residues 214/5–223/4 at m/z 1130.5468 (in which the cysteine residue at position 214/5 is modified) in addition to a unique 1a indicator peptide at m/z 960.4555 (comprising E2 residues 206–213). The strain is thus confidently assigned to be of the 1a subtype. To establish the ability of the proteotyping approach to subtype an uncharacterised strain directly from the proteolysis of a whole virus, the MALDI mass spectrum for such a digest was recorded (Fig. 4). Among the peptide ions detected, the detection of a signature peptide at m/z 1266.8019 (bolded and underlined in the spectrum) corresponding to residues 180–192 of the E1 protein, confirms that the strain is of the 2b subtype. For the purposes of these proteotyping experiments, the remaining spectral information, which represents peptides derived from multiple viral proteins that are not easily interrogated by mass fingerprinting approaches, is superfluous. 4. Discussion

1 Some regions, such as the 5′-UTR or core regions, are well-conserved across genotypes, while others, especially within the E1 and E2 coding regions, are far more diverse.

Despite the widespread use and availability of PCR methodologies 22

Clinical Mass Spectrometry 4–5 (2017) 19–24

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Fig. 3. MALDI mass spectrum of the tryptic digest products of recombinant E2 protein of the 1a subtype.

Fig. 4. MALDI mass spectrum of the tryptic products from a whole virus digest of unknown subtype.

proteotyping approach, to eliminate ambiguities in mass prediction, can be supplemented by other mass spectrometric approaches that allow for the investigation of such glycoforms [30]. The design of the MALDI target plate makes it possible for mass spectra to be acquired from hundreds of digested virus samples within minutes, while the time for virus pretreatment and proteolytic digestion can be minimized by using immobilized enzymes at higher enzyme-to-protein ratios, and multiplexed sample processing. Although a high resolution mass spectrometer poses a considerable cost barrier (AUD $500,000–1,000,000), it is not inconsistent with the costs required for multiple PCR

an alignment of the amplified sequence with reference sequences. Even employing optimal manipulation and handling procedures, viral levels at or below 104 copies represent a limitation to successful PCR amplification and sequencing with operating cost ranging from AUD $1 to 10 per megabase (Mb). In contrast, the proteotyping MS approach requires no separation of viral components when applied to a whole virus digest. The pretreatment (i.e., chemical modification and proteolysis) of samples can be expedited and performed in a time frame similar to the steps necessary for PCR-based methods. The need to deglycosylate viral proteins for the 23

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sequencers and associated equipment. Sensitivities, which have been demonstrated to be on the order of 105–106 copies [24], could be substantially improved by adopting automated single ion monitoring (SIM) strategies that seek to detect only signature/indicator peptides rather than record full mass range spectra. A sensitivity comparison between proteotyping and PCR experiments awaits a side-by-side analysis for common strains from the same set of clinical specimens. Unlike other strategies for peptide and protein identification, proteotyping does not require the use of tandem mass spectrometry (i.e., MS/MS), the use of which adds acquisition and analysis time, and increases sample consumption and minimum sample limits by at least an order of magnitude. The ability of the MALDI target to be coupled to automated, or semi-automated, spectral acquisition instrumentation will facilitate high throughput sample analysis (i.e., seconds per spectrum).

[8]

[9] [10]

[11]

[12]

[13]

5. Conclusions This study demonstrates the ability of a proteotyping approach employing the power of high resolution mass spectrometry to subtype HCV, more directly, through the detection of predetermined signature peptides within whole protein or virus digests. Although illustrated for the more common subtypes of the virus, the approach should be capable of detecting less prevalent subtypes in the population, provided sufficient consensus segments exist that define these subtypes. Additionally, this approach is not challenged by mixed infections where multiple signatures for different subtypes would be detected. The study demonstrates the broader applicability of a single-ion detection proteotyping approach beyond those previously reported for respiratory viruses [18–24]. It offers an alternate method that could be implemented in a clinical diagnostic laboratory, and replace classical PCRbased sequencing methods, in the near future.

[14]

[15]

[16]

[17] [18]

[19] [20]

Conflict of interest statement

[21]

The authors declare there is no conflict of interest.

[22] [23]

Acknowledgements

[24]

R. Uddin was supported in part by an Australian Research Council Discovery Project grant (DP140100591) awarded to K. Downard. K. Downard thanks Prof. Tony Malic and Dr. Berin Boughton for access to the SolariX FT-ICR mass spectrometer at the University of Melbourne.

[25]

[26]

Appendix A. Supplementary data [27]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.clinms.2017.08.003. [28]

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